The project presents a new collaborative research initiative between Oslo University Hospital, Latvian Biomedical Research and Study Centre and Latvian Institute of Organic Synthesis. Our main objective is to perform preclinical evaluation of a novel therapeutic strategy against cancer cells based on combination of chemotherapeutic drugs with viral gene therapy vectors. We propose to apply novel alphaviral vectors encoding cytokines, which able to stimulate tumor-associated macrophage (TAM) infiltration into the tumor and induction of pro-inflammatory microenvironment to block tumor recovery after chemotherapy. The outcomes of treatment will be monitored using advanced imaging technologies including bioluminescent and fluorescent imaging. The project will contribute to research excellence both in Latvia and Norway and will stimulate the development of innovative therapies urgently needed to improve the standard therapeutic approaches for cancer.

Information published: 05.05.2015.

May-July 2015

12.05.2015 project kick-off teleconference

05.06.2015 project seminar at LIOS

Development of liposomal nanoparticles for delivery of therapeutic genes and chemotherapeutic drugs is started

Construction and synthesis of alphaviral DNA vectors is started

Development of human PDX tumor models is started

August-October 2015

First liposomal compounds and fluorescent dyes were synthesised

New alphaviral vectors producing anti-cancer citokines were constructed and synthesized

New fluorescent dyes were tested for the ability to stain cancer cell cultures

Macrophage polarization experiments have started

SUMMARY OF PROJECT ACTIVITIES (May-December, 2015)

Within the first reporting period, the involved three groups of scientists from Latvian Biomedical Research and Study Centre (LBMC), Latvian Institute of Organic Synthesis (LIOS) and Oslo University Hospital (OUH) have finished the project activities devoted to establishment of tumor models, synthesis of drug candidates and potent cancer gene therapy vectors. The Tumor Immunology group headed by Dr. Corthay (OUH) has finalized the first detailed characterization of the “Immune Cell Composition in Human Non-small Cell Lung Cancer” (a manuscript is submitted for publication). This knowledge will serve as a basis for the planned immunotherapy experiments. The group has also established the patient-derived xenotransplantation (PDX) mouse model for lung cancer that will be used to test immunotherapy protocols with alphaviruses.

At the same time, the Latvian Cancer Gene Therapy group headed by Dr. Zajakina (LBMC) has produced first alphaviral vectors encoding therapeutic gene candidates for reprogramming of tumor microenvironment to neglect the immune tolerance to cancer cells in breast cancer and lung cancer models. Novel constructs for expression of mouse and human interferon gamma and tumor necrosis factor alpha will be used in combination with advanced chemotherapeutics synthesized by the group of Prof. Duburs (LIOS). Series of putative delivery agents on the basis of 1,4-dihydropyridine (1,4-DHP) core were obtained by a variation of different alkyl moieties at the 3,4 and 5 positions of 1,4-DHP cycle (a manuscript is submitted for publication). Moreover, synthesis and characterization of fluorescent compounds comprising electron donor groups and conjugated electron accepting moieties was performed in order to obtain polychromic probes possessing near infrared (NIR) emission for efficient fluorescent imaging and drug tracking in vivo.

Furthermore, LBMC group together with OUH colleagues have finished experiments related to proteome analysis of B16 mouse melanoma cells susceptible and unsusceptible to alphavirus infection. These results pave the way for optimized development of strategies for designing tumor tropism of alphaviral vectors as potent cancer gene therapy tool. All together the first project period has shown the successful implementation of the proposed research ideas and fruitful results of the complementary collaboration between the partners.

February - April 2016

It is known that interferon-γ (IFN-γ) primes macrophages for enhanced inflammatory activation and acquisition of M1 phenotype by Toll-like receptors (TLRs). As M1 macrophages have anti-tumorigenic and tumoricidal functions they could be used in cancer immunotherapy. Whereas tumour necrosis factor-a (TNFa) has been suggested to have cancer-suppressive properties as well. Though TNFa is a potential therapeutic for cancer immunotherapy, targeted delivery of TNFa to tumours is needed to limit its toxicity.

In order to investigate the potential of cytokine-expressing Semliki Forest virus (SFV) based vectors in cancer immunotherapy, three new SFV vector based alphaviral constructs incorporating mTNFa, mIFNg and hIFNg genes were developed. SFV based vectors were shown to infect Lewis Lung carcinoma (LLC) cells and greatly inhibit their growth in vitro. In order to study whether SFV-mIFNg derived IFNg could enhance macrophage activation through TLRs in vitro, bone marrow derived macrophages (BMDM) were challenged with SFV-mIFNg derived mIFNg and effect on LLC cell growth was detected. SFV derived mIFNg functionality in vitro was shown in two independent experiments. It was concluded that SFV derived mIFNg could be used for enhanced activation of macrophages to cancer suppressive phenotype through different TLRs.

Future in vivo experiments comprises the use of SFV-mTNFa for cancer growth inhibition in combination with SFV-mIFNg whereas SFV-hIFNg construct will be used on human lung cancer xenotransplantant model in vivo.

May - July 2016

The involved three groups of scientists from Latvian Biomedical Research and Study Centre (LBMC), Latvian Institute of Organic Synthesis (LIOS) and Oslo University Hospital (OUH) continued the project implementation activities according to the work plan. The synthesised drug candidates (LIOS) and viral vectors (LBMC) were tested in pilot experiments using in vitro and in vivo cancer models established at OUH. The primary results indicate that Semliki Forest virus (SFV) vectors expressing macrophage stimulating cytokines (produced at LBMC) can activate tumor associated macrophages to kill cancer cells in presence of specific TLR ligands. The treatment with SFV vectors alone or together with TLR ligands was investigated in tumor growth inhibition experiments (OUH). The pilot results have demonstrated positive therapeutic outcome of the proposed treatment strategy.

Furthermore, the LIOS synthesised fluorescent compounds showed a high potential to be used as cancer cell tracking agents in vivo in mice models. These novel fluorescent compounds possessing fluorescent properties within the infrared excitation spectrum can be further developed as theranostic drugs for cancer diagnostic and therapy.

In frame of the project, three international pier reviewed articles were published:

Within the third reporting period, the involved three groups of scientists from Latvian Biomedical Research and Study Centre (LBMC), Latvian Institute of Organic Synthesis (LIOS) and Oslo University Hospital (OUH) continued the project implementation activities according to the work plan. The synthesised fluorescent drug candidates (LIOS) were tested in vitro and in vivo in mouse models established at OUH. The primary results indicate that fluorescent compounds can be visualized in vivo in infrared emission spectrum. Furthermore, several synthetic compounds, derivatives of 1,4-DHP, were selected for cancer treatment due to their polyfunctional properties which include high antiproliferation and gene delivery potential. These drugs will be tested in Lewis lung carcinoma model in combination with alphaviral vectors developed by LBMC.

Within the reporting period two papers were published/accepted for publication:

The project contributes to development of new anti-cancer therapies based on alphaviral vectors expressing immunomodulating cytokines and 1,4-dihydropiridine-based chemotherapeutics with fluorescent properties. The main idea of the project was to design viral vectors for activation of tumorocidal functions of tumor assotiated macrophages, which will be applied in combination with chemical drugs possessing antiproliferative as well as infrared-spectrum emission characteristics.

Several recombinant Semliki Forest (SFV) and Sindbis (SIN) virus vectors encoding either tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) were developed. The vectors showed high infection rate and cytotoxicity in mouse and human cancer cells in vitro (lung and breast cancer models). By contrast, mouse and human macrophages were resistant to infection with SFV. The recombinant SFV vectors directly inhibited mouse lung carcinoma cell growth in vitro, while exploiting the cancer cells for production of SFV vector-encoded cytokines. The functionality of SFV vector-derived TNF-alpha was confirmed through successful induction of cell death in TNF-alpha-sensitive fibroblasts in a concentration-dependent manner. The SFV vector-derived IFN-gamma activated macrophages toward a tumoricidal M1 phenotype leading to suppressed lung carcinoma cell growth in vitro. The ability of SFV to provide functional cytokines and to infect tumor cells but not macrophages suggests that SFV may be very useful for cancer immunotherapy employing tumor-infiltrating macrophages.

In a frame of development of novel chemotherapeutics, three potential anti-cancer drugs were selected for combined treatment with alphaviral vectors. The compounds demonstrated inhibition of Lewis lung carcinoma growth through induction of necrosis of tumor nodules, as was confirmed by histochemical analysis of tumors. Fluorescent properties of the compounds were evaluated using microscopy and in vivo imaging system. More than ten compounds and linkers were designed and synthesized in order to improve their tumor targeting properties and fluorescence characteristics. The next step will include the attachment of tumor homing ligands to the most promising compounds.

Furthermore, a mouse model based on patient derived tumor xenografts was developed and comprehensively characterised in terms of immune cell composition and growth dynamics. The model allows evaluation of therapeutic effects in presence of human immune cells, which are transplanted within the xenograft pieces.

In summary, although the obtained results were fruitful, the implemented project represents a beginning of collaborative research initiative between Latvia and Norway in the field of cancer therapy. The involved groups with complementary expertize have a clear vision of sustainable collaboration. Practically, next steps will include further development of anti-cancer drugs, evaluation of 1,4-dihydropiridine-based drugs in PDX models, activation of human macrophages with alphaviral vectors and optimization of vector delivery mode in mouse allograft and human xenograft models.

The project resulted in six articles in international pier-reviewed journals (four published, one accepted and one submitted for publication), two of them are joint publication.